The enzyme ACC synthase is essential to the production of ethylene in higher plants. It is well known that ethylene is related to various events in plant growth and development including fruit ripening, seed germination, abscission, and leaf and flower senescence. Ethylene production is strictly regulated by the plant and is induced by a variety of external factors, including the application of auxins, wounding, anaerobic conditions, viral infection, elicitor treatment, chilling, drought, and ions such as cadmium and lithium ions. A review of ethylene production and effects in plants may be found, for example, in Abeles, F. B., "Ethylene in Plant Biology" (1983) Academic Press, New York.
It is also known that the synthesis of ethylene in higher plants includes a rate limiting step which is the conversion of S-adenosyl methionine (AdoMet) to 1-aminocyclopropane-1-carboxylic acid (ACC). This conversion is catalyzed by the enzyme ACC synthase (EC4.4.1.14). This enzyme has been partially purified from several sources by Nakajima, N., et al., Plant Cell Physiol (1986) 27:969-980; Mehta, A. M., et al., Proc Natl Acad Sci USA (1988) 85:8810-8814; Nakajima, N., et al., Plant Cell Physiol (1988) 29:989-990; Tsai, D. S., et al., Arch Biochem Biophys (1988) 264:632-640; Bleecker, A. B., et al., Proc Natl Acad Sci USA (1986) 83:7755-7759; Privale, L. S., et al., Arch Biochem Biophys (1987) 253:333-340; Sato, S., et al., Plant Physiol (1988) 88:109-114; Van Der Straeten, D., et al., Eur J Biochem (1989) 182:639-647.
As the level of ACC synthase controls the production of ethylene, control of the level of this enzyme permits control of ethylene levels and thus regulation of the plant growth and development aspects that are controlled by ethylene. The availability of the relevant ACC synthase expression system and coding sequences permits control of ACC synthase expression and activity, as provided by the invention herein.
In an abstract published in connection with the UCLA Symposia on Molecular and Cellular Biology, held Mar. 27-Apr. 7, 1989, as published in J Cell Biochem (1989) Supp. 13D, page 241, Theologis, A., et al. disclosed that a cDNA sequence designated pACC1 from Cucurbita (zucchini) fruits had been isolated by screening a cDNA library in .lambda.gt11 with antiserum prepared by subtraction purification using proteins obtained from tissues that were induced and uninduced for ACC synthase. The pACC1 clone was reported to hybridize to a 1900 nucleotide mRNA that was induced by auxin and lithium ions. The abstract further reports that using the Cucurbita cDNA as a probe, cDNA and genomic clones encoding tomato ACC synthase were isolated, and that the authenticity of these clones had been confirmed by recovery of enzyme activity after expression in E. coli. An expanded version of the work described in this abstract was published by Sato, T. et al., Proc Natl Acad Sci USA (1989) 86:6621-6625. However, neither the abstract nor the paper disclosed the details of the purification of the native ACC synthase. An additional abstract further reporting this work and indicating that the ACC synthase in tomatoes was encoded by a single-copy gene was published in connection with the succeeding UCLA Symposium on Molecular and Cellular Biology in J Cell Biochem (1990) Supp. 14E, page 358. This symposium was held Mar. 31-Apr. 22, 1990.
Two additional accounts of the recovery of cDNA encoding the ACC synthase of Cucurbita fruit, and further indicating that the Cucurbita genome contains two linked ACC synthase genes which are transcribed in opposing directions were published in Horticultural Biotechnol (1990) Wiley-Liss, Inc., pp. 237-246 and in Plant Gene Transfer (1990) Alan R. Liss, Inc., pp. 289-299. A further summary was presented in an abstract published in connection with Plant Molecular Biology Meeting conducted by the NATO Advanced Study Institute held in Bavaria on May 14-23, 1990 and at the Third International Symposium of the Society of Chinese Bioscientists in America, held Jun. 24-30, 1990. None of these publications disclosed the nucleotide sequences of either the coding or control regions for any of the ACC synthase genes.
Sato, T. et al., J Biol Chem (1991) 266:3752-3759 described the isolation, properties, and expression in E. coli of the 50 kd ACC synthase of Cucurbita as encoded by the cDNA prepared from messenger RNA present in fruits induced with auxin and lithium ion. The preparation of this cDNA and the complete deduced amino acid sequence thereof are described hereinbelow in Example 1 and FIG. 1.
Van der Straeten, D., et al. reported the cloning and sequences of cDNAs purportedly encoding ACC synthase from tomato (Proc Natl Acad Sci USA (1990) 87:4859-4863). Although the cDNA, which corresponded to an open reading frame of approximately 55 kd, produced a 55 kd peptide in E. coli, the authors were unable to show ACC synthase activity in the extracts of E. coli producing this protein. Comparison with the sequences described in the present invention shows that two amino acid residues that are invariant among the polypeptides found herein are miscoded in the vector reported by van der Straeten; specifically, Leu322 was changed to Pro322 and Pro399 was changed to Leu399. It is believed that these changes in the highly conserved regions lead to inactivated forms of this protein.